PROJECT SUMMARY Neuroendocrine and metabolic stressors threaten cellular and organismal integrity, leading to maladaptive cellular changes and disease unless met by adaptive remodeling of nuclear gene expression. Mitochondria are central to these adaptations. Recent findings indicate that mitochondria participate in a three- step intracellular signal transduction system involving sensing, signal integration and transduction to the nucleus where they regulate the majority of genes within the human genome. In this way, mitochondria are a emerging as determinants of cellular and organismal adaptation to common stressors. The overall objective of this proposal is to define novel mechanisms of mitochondria-mitochondria and mitochondria-nuclear signaling leading to gene expression remodeling. To achieve this, my laboratory uses use drug-inducible inter-organellar linker technology to manipulate mito-mito and mito-nuclear interactions in muscle cells, coupled to high-resolution quantitative light and electron microscopy approaches to track organelle interactions. We exploit high-throughput functional assays, metabolomics, and transcriptomics to visualize and understand the resulting nuclear transcriptional responses patterns to stressors. To disentangle the relative contributions of mitochondrial network organization and functions to mitochondrial signaling, we leverage unique trans-mitochondrial cell and animal models, as well as mitochondria-targeted small molecule antioxidants and pharmacological agents. Candidate signaling pathways will be validated using parallel genetic and biochemical experiments. Most promising pathways will be extended in follow up studies using a near- experimental human disease model of primary mitochondrial DNA defects to validate our findings in humans. Together, this combined approach will investigate specific components of the mitochondria-nuclear communication system and their relevance to human disease. This work will establish the physical basis for gene expression regulation by mitochondria, and serve as the foundation for further work aiming to circumvent maldaptative cellular and organismal responses to stressors and mitochondrial dysfunction.